The 3-Phosphoinositide Dependent Protein Kinase-1 (PDK1) is a key protein kinase, regulating activity of a group of related protein kinases through phosphorylation. These kinases include isoforms of Protein Kinase B (also known as Akt) [Brazil and Hemmings, 2001, Scheid and Woodgett, 2001], p70 ribosomal S6 kinase (S6K) [Alessi et al., 1997, Volarevic and Thomas, 2001], p90 ribosomal S6 Kinase (RSK) [Frodin and Gammeltoft, 1999] and the serum and glucocorticoid induced-protein kinase (SGK) [Lang and Cohen, 2001]. These enzymes are stimulated by hormones and growth factors and phosphorylate regulatory proteins mediating the various physiological effects of these agonists.
PDK1 possesses an N-terminal kinase catalytic domain and a C-terminal pleckstrin homology (PH) domain [Alessi et al., 1997, Stephens et al., 1998]. PDK1 activates its substrates by phosphorylating these kinases at their activation loop (reviewed in [Alessi, 2001, Toker and Newton, 2000]). The phosphorylation of PKB by PDK1 is dependent upon prior activation of the phosphoinositide 3-kinase (PI-3-kinase) and the production of the second messenger, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) which binds to the PH domains of PDK1 and PKB. This does not activate either PKB or PDK1 but instead recruits and co-localises these enzymes at the plasma membrane.
Unlike PKB, the other PDK1 substrates described thus far do not interact with PtdIns(3,4,5)P3 nor is the rate at which they are phosphorylated by PDK1 further enhanced by the binding of PDK1 to PtdIns(3,4,5)P3. Instead the ability of PDK1 to phosphorylate S6K, SGK and RSK is promoted by phosphorylation of these enzymes at a residue located C-terminal to the kinase catalytic domain in a region known as the hydrophobic motif [Alessi et al., 1997, Kobayashi and Cohen, 1999, Pullen et al., 1998]. The kinases that phosphorylate the hydrophobic motif of S6K and SGK are unknown but as the phosphorylation of this residue in vivo is dependent on PI-3-kinase activation, the hydrophobic motif kinases and/or the hydrophobic motif phosphatases may be regulated by PtdIns(3,4,5)P3. In the case of RSK isoforms, phosphorylation by the ERK1/ERK2 MAP kinases induce phosphorylation of the hydrophobic motif (reviewed in Frodin and Gammeltoft, 1999).
PDK1 belongs to the same subfamily of protein kinases as its substrates, termed the AGC protein kinases as they are related to the cAMP dependent protein kinase (PKA)/cGMP dependent protein kinase/Protein kinase C (PKC). PKA is the only AGC kinase whose crystal structure has been solved. Like all protein kinases, its catalytic core possesses an N-terminal lobe consisting mainly of β-sheet and a predominantly α-helical C-terminal lobe [Taylor et al., 1992, Husen and Kuriyan, 2002]. The ATP binding site is located in between the 2 lobes [Johnson et al., 2001, Knighton et al., 1991]. At the very C-terminus, PKA possesses an extended loop that terminates in the sequence FXXF (SEQ ID NO:1) which resembles the first part of the hydrophobic motif phosphorylation site of S6K and SGK (FXXFS/TY, SEQ ID NO:2) in which the Ser/Thr is the phosphorylated residue [Biondi et al., 2000]. In the structure of PKA, the FXXF motif (SEQ ID NO:1) is buried in a hydrophobic pocket in the small lobe of the PKA catalytic domain [Knighton et al., 1991] and mutation of either of the Phe residues drastically reduces PKA activity towards a peptide substrate [Etchebehere et al., 1997]. Unlike other AGC kinases, PDK1 does not possess a hydrophobic motif C-terminal to its catalytic domain. However, there is evidence that PDK1 possesses a hydrophobic pocket in the small lobe of its catalytic domain similar to that in PKA. We have biochemically demonstrated that the interaction of PDK1 with four of its substrates (S6K1, SGK1, PKζ and PKC related kinase-2 (PRK2)) is reduced or abolished by mutation of residues predicted to form part of this pocket [Balendran et al., 2000, Biondi et al., 2000]. Furthermore, mutation of a central residue in the predicted pocket, Leu 155, prevented PDK1 from phosphorylating and activating S6K1 and SGK1 without affecting its ability to phosphorylate either PKB or a short peptide substrate that encompasses the activation loop of PKB (T308tide) [Biondi et al., 2000]. The hydrophobic pocket on the kinase domain of PDK1 has been termed the “PIF-pocket” after the name of the first AGC-kinase hydrophobic motif-containing peptide (PDK1 Interacting Fragment) that was found to bind PDK1 [Balendran et al., 1999a]. It has been suggested that the PIF-pocket in PDK1 functions as a docking site, enabling PDK1 to interact with some of its physiological substrates. Furthermore, there is evidence that phosphorylation of the hydrophobic motif of S6K1, SGK and RSK2 [Balendran et al., 1999b, Biondi et al., 2001, Frodin et al., 2000] promotes the interaction of these enzymes with PDK1. These findings suggest that the PIF-pocket on PDK1 could contain a phosphate binding site promoting the binding of PDK1 to a subset of substrates (S6K, SGK and RSK) once these enzymes have been phosphorylated at their hydrophobic motif. This would result in a physiological phosphate dependent interaction. In addition there is evidence that occupancy of the PIF-pocket activates PDK1 as peptides that encompass the hydrophobic motif of PRK2 [Biondi et al., 2000] and RSK [Frodin et al., 2000] induce a 4-6-fold activation of PDK1.
Previous predicted structures PDK1 catalytic domain were obtained using homology modelling methods based upon structural information available from the catalytic domain of PKA (Biondi et al., 2000). These predictions of the PDK1 catalytic domain structure were thus biased towards the catalytic domain from which the structural information was obtained.